This invention relates generally to an improved apparatus for the performance of chemical processes and, more particularly, to an improved apparatus for automatically performing the sequential degradation of protein or peptide chains containing a large number of amino acid units for purposes of determining the sequence of those units.
The linear sequence of the amino acid units in proteins and peptides is of considerable interest as an aid to understanding their biological functions and ultimately synthesizing compounds performing the same functions. Although a variety of techniques have been used to determine the linear order of amino acids, probably the most successful is known as the Edman Process. Various forms of the Edman Process and apparatuses for automatically performing the processes are described in the following publications:
Edman and Begg, "A protein Sequenator," European J. Biochem. 1 (1967) 80-91; Wittman-Liebold, "Amino Acid Sequence Studies of Ten Ribosomal Proteins of Escherichia coli with an Improved Sequenator Equipped with an Automatic Conversion Device," Hoppe-Seyler's Z. Physiol. Chem. 354, 1415 (1973); Wittmann-Liebold et al., "A Device Coupled to a Modified Sequenator for the Automated Conversion of Anilinothiazolinones into PTH Amino Acids," Analytical Biochemistry 75, 621 (1976); Hunkapiller and Hood, "Direct Microsequence Analysis of Polypeptides Using an Improved Sequenator, A Nonprotein Carrier (Polybrene), and High Pressure Liquid Chromatography," Biochemistry 2124 (1978); U.S. Pat. No. 3,725,010 issued to Penhasi on Apr. 3, 1973, for "Apparatus for Automatically Performing Chemical Processes;" and U.S. Pat. No. 3,717,436 issued to Penhasi et al. on Feb. 20, 1973, for "Process for the Sequential Degradation of Peptide Chains." Briefly, as discussed in the above publications, the Edman sequential degradation processes involve three stages: coupling, cleavage, and conversion. In the coupling stage phenyl isothiocyanate reacts with the N-terminal .alpha. amino group of the peptide to form the phenylthiocarbamyl deriviative. In the cleavage step anhydrous acid is used to cleave the phenylthiocarbamyl derivative, i.e., the anilinothiazolinone. After extraction of the thiazolinone the residual peptide is ready for the next cycle of coupling and cleavage reactions. Aqueous acid is used to convert the thiazolinone to the phenylthiohydantoin which may be analyzed in an appropriate manner such as by chromatography.
The automated apparatus of the Penhasi U.S. Pat. No. 3,725,010 patent, as modified in the above-referenced articles of Wittmann-Liebold and the article of Hunkapiller and Hood, is the most sophisticated prior sequenator known to us. The reactions in this sequenator are carried on in a thin film formed on the inside wall of a rotating reaction cell located within a closed reaction chamber. Means are provided for introducing and removing liquids and gases relative to the chamber, and the reaction cell is substantially closed off from the reaction chamber such that liquids in the reaction cell are substantially trapped therein but gases fed into the reaction chamber may enter and circulate in the reaction cell. The protein or peptide being analyzed is initially placed in the rotating reaction cell, followed by the sequential introduction and withdrawal of the various reagents and solvents necessary for carrying out the coupling and cleavage reactions. Upon completion of the cleavage step, the resulting thiazolinone is extracted and transferred either to a separate flask for conducting the conversion step or to an apparatus for collection and drying of the various fractions. In cases where the conversion process is not performed immediately in a conversion flask, the process may be performed later on a number of fractions simultaneously.
The introduction and withdrawal of fluids relative to the reaction cell has generally been achieved with fluid conduits passing through a plug which seals an opening in the upper wall of the reaction chamber and depends therefrom to a location within the reaction cell. Fluids are introduced directly into the reaction cell at a point adjacent the bottom thereof, and are withdrawn from an annular groove in the cylindrical interior surface of the reaction cell. The fluid to be withdrawn is forced into the annular groove by centrifugal force when the cup is rotated at a high rate, and is withdrawn through a conduit having an inner end projecting into the groove. This effluent conduit thus acts as a scoop for removing the reaction products and by-products and the extracting solvents from the reaction cell. Because the orientation of the inner end of the effluent line relative to the groove and the reaction cell is critical to the performance of the scooping operation, the effluent lines have been constructed to be externally adjustable in extension and angular orientation relative to the plug. The degradable elastomers and other materials associated with such adjustable fittings have caused contamination of the sample, both through reaction with the chemicals in the system and the production of leaks to the atmosphere.
Drying of the various reagents and solvents at the appropriate times is produced in part by vacuum means. It is generally desirable to vacuum dry the sample in two steps to avoid disruption of the system by boiling the more volatile components. A restricted vacuum is thus first applied to the system to draw out the bulk of the volatile components. The full output of the vacuum pump is then applied to complete the drying operation. Restriction of the vacuum is accomplished through use of a connecting passage having a much more restricted bore than the passage used to achieve full vacuum. The passages are opened and closed by respective solenoid valves kept at room temperature. In this configuration, semivolatile reagents and solvents have a tendency to condense on the bore of the restricted vacuum tube and also within the bodies of the two vacuum valves. It is thus very difficult to fully evacuate the reagents and solvents from these areas at the desired times, contaminating the chemistry of subsequent coupling and cleavage steps.
The prior devices have each used a number of degradable elastomeric seals which can contaminate the system both by leakage and reaction with its components.
The contamination caused by the several features described above has a cumulative effect over the duration of a sequential degradation process. The sample and the reagents within the reaction cell thus become more and more contaminated, hindering the desired coupling and cleavage reactions and causing a number of undesired reactions to take place. The yield from each complete cycle of the apparatus is thus decreased and a series of contaminants are introduced into the fractions.
While these effects may be overlooked in some cases where large amounts of the protein or peptide sample are available or where the chain has a relatively small number of units, they become devastating in cases where the chain has a very large number of units or only very small amounts of the particular protein or peptide are available. Both of these circumstances are present in the case of interferon, a small protein made in human cells in response to certain viral infections. Interferon has recently caused a great deal of excitement in the world of clinical medicine because it promises to be an effective agent for arresting viral infections and it appears to offer considerable hope as an anti-cancer reagent. Interferon is produced and, accordingly, is available only in very small quantities. Currently, virtually the entire world's production of the two types of human interferon originates in the relatively few world centers that have access to large quantities of human white blood cells (leukocyte interferon) or certain human cells in tissue culture (fibroblast interferon). Because of this limited productive capacity for interferon, it has been difficult to carry out well controlled clinical studies and fundamental analyses of how this molecule functions. To further complicate the picture, interferon is composed of a chain of approximately 150 amino acid residues, which must be individually cleaved from the chain for analysis. The contamination losses inherent in the operation of the prior sequenators have thus far prevented the sequencing of any but the first few amino acid units of interferon with the very small available quantities of the protein. Beyond the first few cleavage cycles, the small sample becomes contaminated to the point at which positive results are unobtainable.
Therefore, in many applications, it is desirable to provide an apparatus for performing chemical processes such as the sequencing of interferon which maintains the sample and everything coming in contact with the sample as free of contamination as possible to enable the maximum number of sequencing cycles to be successfully performed with the minimum amount of sample.